Evidence against Zika virus infection of pets and peri-domestic animals in Latin America and Africa

Decades after its discovery in East Africa, Zika virus (ZIKV) emerged in Brazil in 2013 and infected millions of people during intense urban transmission. Whether vertebrates other than humans are involved in ZIKV transmission cycles remained unclear. Here, we investigate the role of different animals as ZIKV reservoirs by testing 1723 sera of pets, peri-domestic animals and African non-human primates (NHP) sampled during 2013–2018 in Brazil and 2006–2016 in Côte d'Ivoire. Exhaustive neutralization testing substantiated co-circulation of multiple flaviviruses and failed to confirm ZIKV infection in pets or peri-domestic animals in Côte d'Ivoire (n=259) and Brazil (n=1416). In contrast, ZIKV seroprevalence was 22.2% (2/9, 95% CI, 2.8–60.1) in West African chimpanzees (Pan troglodytes verus) and 11.1% (1/9, 95% CI, 0.3–48.3) in king colobus (Colobus polycomos). Our results indicate that while NHP may represent ZIKV reservoirs in Africa, pets or peri-domestic animals likely do not play a role in ZIKV transmission cycles.

The Zika virus (ZIKV) was first isolated from a sentinel rhesus monkey (Macaca mulatta) in Uganda during the 1950s and later reported in native non-human primate (NHP) species and in forest-dwelling Aedes mosquitos in Africa and therefore transmission in parts of Africa is thought to be maintained through interepidemic sylvatic cycles [1,2]. ZIKV was not considered a major threat to human health [3,4] until its emergence in Latin America, which was associated with severe congenital malformations including microcephaly [5]. According to evolutionary reconstructions, ZIKV was likely introduced into Brazil during 2013 [5] and infected approximately 8.5 million people during 2015 and 2016 [6]. Community protective immunity in urban centers likely contributed to cease of the epidemic [7].
Sylvatic transmission cycles may permit ZIKV maintenance until the pool of susceptible humans is replenished by birth and migration, potentially contributing to a resurge of intense urban ZIKV transmission cycles. Whether animal reservoirs play a role in ZIKV transmission in the Americas remains unknown. The available data on ZIKV infection of NHP in the Americas are inconclusive. Some South American NHP species, such as marmosets (Callithrix jacchus) and tamarins (Saguinus labiatus) were susceptible to ZIKV in vivo [8]. Low seroprevalence and low levels of neutralizing antibodies have been reported in capuchin OPEN ACCESS monkeys in Northeastern and Central Brazil [9,10]. Finally, ZIKV has been detected in NHP carcasses in the Southeastern region, but since contamination of the remains could not be excluded, the relevance of those findings still needs to be confirmed [11].
Several flaviviruses can infect pets and peri-domestic animals. For instance, experimental infection and serological evidence indicate circulation of West Nile virus (WNV) and Japanese Encephalitis virus (JEV) in cats and dogs [12][13][14][15] and of Wesselsbron virus (WSLV) in cattle, goat and sheep [15][16][17][18][19][20][21]. Virus detection by molecular methods, virus isolation and serological evidence substantiate that horses can be infected with WNV, Saint Louis Encephalitis virus (SLEV), JEV and Usutu virus (USV) [22,23]. Livestock species, such as sheep and goats, were susceptible to ZIKV in vivo [24,25], and although ZIKV-specific neutralizing antibodies were reported in sero-epidemiological studies in cattle and sheep from Brazil and in horses from French Pacific Islands [26,27], only few flaviviruses beyond ZIKV were used in those studies to rule out potentially unspecific test results elicited by cross-reactive flavivirus antibodies. Exposure of vertebrates to ZIKV depends on the feeding preferences of the mosquito vectors. In Latin America, ZIKV vectors include predominantly Aedes aegypti and, to a lesser extent, Ae. albopictus [2,28,29]. In Africa, the invertebrate host range of ZIKV is not entirely known. However, major vectors likely include Aedes aegypti formosus, Ae. africanus, Ae. albopictus, Ae. apicoargenteus, Ae. furcifer and Ae. vitattus [28]. Whereas Ae. aegypti and, to a lesser extent, Ae. albopictus are highly anthropophilic, they are also known to opportunistically feed on other vertebrates, including rodents, cattle, sheep and dogs [29][30][31][32][33][34]. Those animals are reared in high numbers and in close proximity to humans and may thus be important components of (peri-)urban ZIKV transmission cycles. Therefore, it is currently unclear whether pets and peri-domestic animals play a role in the maintenance of ZIKV in Africa or the Americas.
Here we investigated ZIKV spread in pets (i.e. dogs and cats) and peri-domestic animals (i.e. equids, cattle, sheep and goats) sampled in northeastern Brazil, the 2015-2016 outbreak's epicentre, and in wild NHP, pets and peri-domestic animals sampled in Côte d'Ivoire, where ZIKV has been documented in forest-dwelling mosquitos [4,35].
In Brazil, equid, cattle, sheep, goat, dog and cat samples were collected in the northeastern state of Bahia during 2013-2018 within state routine veterinary surveillance activities or from dogs and cats presenting clinical signs, and in 2015 from dogs in the neighbouring state of Pernambuco for a Leishmania spp. serosurvey [36]. In Côte d'Ivoire, peri-domestic animals were sampled in 2012 and 2014. Unhabituated wild NHP such as king colobus (Colobus polycomos) and western red colobus (Piliocolobus badius) were sampled in the Taï National Park during 2006 and 2016 [37,38]. Samples were also collected from a habituated group of sooty mangabeys (Cercocebus atys) that has been under observation since 2012 [37,38]. Additionally, nine blood samples from deceased West African chimpanzees (Pan troglodytes verus) were included; this population of chimpanzees has been followed daily since 1979 and necropsy samples have been systematically performed on all dead individuals recovered, since the inception of the veterinary programme in 2002 [37,39].
Antigenic cartography did not provide a robust separation of flavivirus serocomplexes in pets and peri-domestic animals from either Côte d`Ivoire or Brazil (Fig. 2d). The detection of sera with monotypic reaction or titres ≥fourfold for SPOV, and WSLV in Brazilian animals must be carefully interpreted. WSLV has not been reported in the Americas, and although SPOV has been isolated in Culex quinquefasciatus in Haiti [44] it is unlikely that either SPOV or WSLV would be widely dispersed among pets or peri-domestic animals from Brazil because there are no robust data on isolation or molecular detection of those viruses in vertebrates from Latin America. A more plausible explanation for those PRNT results are cross-reactive flavivirus antibodies, which complicate the interpretation of flavivirus serological assays in hyperendemic settings, even when considering only ≥fourfold PRNT 90 titres as decisive serological support [42,45]. This interpretation is supported by the overall low titres against all tested flaviviruses and by previous evidence showing the difficulties to confirm human ZIKV infection in DENV-endemic areas [42,46].
In NHP, ZIKV ECLIA reactivity was observed in 23.1% (3/13, 95% CI, 11.1-39.3) of red colobus, 66.7% (6/9, 95% CI, 29.9-92.5) of king colobus and in none of the 17 sooty mangabeys, while 88.9% (8/9, 95% CI, 51.8-99.7) of the chimpanzees tested positive for ZIKV AF using PRNT 90 . Fourfold or higher ZIKV PRNT 90 titres compared to other flaviviruses were found in 11.1% (1/9, 95% CI, 0.3-48.3) of king colobus and in 22.2% (2/9, 95% CI, 2.8-60.1) of chimpanzees (Table 1, Fig. 2c, e) and in none of the previously ECLIA-reactive red colobus. Moreover, one chimpanzee (1/9, 95% CI, 0.3-48.3) presented titres ≥fourfold for WNV compared to all other flaviviruses. The difference in NHP ZIKV seroprevalence might be related to differential host susceptibility or different ecological niches occupied by those NHP since the mosquito species from which ZIKV was isolated in Côte d'Ivoire (i.e. Aedes vitattus, Ae. furcifer and Ae. aegypti formosus) are predominantly found in the tree canopy [28,35,47]. Notably, ZIKV was linked to NHP already in the initial viral isolation from a sentinel Rhesus macaque caged in the canopy [48]. Our data suggest that some NHP species like chimpanzees and king colobus, which are at least partly arboreal, might play a role as ZIKV reservoirs in Côte d'Ivoire. Our interpretation is supported by previous studies indicating ZIKV exposure of native African NHP by viral isolation from Patas (Erythrocebus patas) and Vervet (Chlorocebus pygerythrus) monkeys in Senegal [49] and by the detection of ZIKV-neutralizing antibodies in different NHP species such as in red-tailed monkey (Cercopithecus ascanius), grey-cheeked mangabey (Lophocebus albigena), mantled guereza (Colobus guereza) in Uganda [50], and tantalus (Chlorocebus tantalus) and Mona (Cercopithecus mona) monkey in Nigeria [51]. On the other hand, our data do not provide conclusive evidence on whether those NHP species may serve as amplification or reservoir hosts, because of our small sample size and because our methods were not targeted towards detecting active infections. Future longitudinal studies including younger NHP, as performed for CHIKV reservoir studies in Senegal [52], could clarify the role of those NHP species for ZIKV transmission in Africa.
Our study was limited by the lack of longitudinal sampling from the different animal species in both locations in Brazil and Côte d'Ivoire, since long-term titre comparison would be more effective for understanding antibody responses after multiple flavivirus infections. We also did not test other synanthropic animals such as marsupials, known to be relevant arbovirus hosts, e.g. for the alphavirus Ross River virus [53] and which may be exposed to ZIKV vectors [54]. However, while most of the previous studies on ZIKV animal reservoirs either used tests with lower specificity only (e.g. ELISA, complement fixation and haemagglutination) or assessed flaviviral cross-reactivity using fewer viruses in PRNT [27,50,51], we provided data from a large number of sera of pets and peri-domestic animals from two continents and performed extensive neutralization testing, considered the gold-standard technique.
In brief, our data confirm that co-circulating flaviviruses challenge unambiguous ZIKV antibody test results in hyper-endemic areas [41,42]. Despite those technical limitations, our data imply that while some NHP species in Côte d'Ivoire may serve as ZIKV reservoir hosts, pets and peri-domestic animals are neither involved in ZIKV transmission cycles in Brazil, nor in Côte d'Ivoire.
Funding information J.F.G. was supported by the Deutsche Forschungsgemeinschaft (DFG) Research Group 'Sociality and Health in Primates' (FOR2136; CA 1108/3-1). This work was supported by the European Union's Horizon 2020 research and innovation programme through the ZIKAlliance project (grant agreement no. 734548) to J.F.D. and X.d.L. and by Roche Diagnostics through the investigator-initiated study ZikaStock to J.F.D.